The present invention relates generally to optical instruments for measuring refractive index of a substance, and more particularly to an optical configuration for measuring a difference in refractive index between first and second samples, for instance a test sample and a reference sample. The present invention is applicable to differential refractometers and surface plasmon resonance (SPR) biosensor devices.
Refractometers measure the critical angle of total reflection by directing an obliquely incident non-collimated beam of light at a surface-to-surface boundary between a high refractive index prism and a sample to allow a portion of the light to be observed after interaction at the boundary. In transmitted light refractometers, light that is transmitted through the sample and prism is observed, while in reflected light refractometers, the light that is reflected due to total reflection at the surface-to-surface boundary is observed. In either case, an illuminated region is produced over a portion of a detection field of view, and the location of the shadowline between the illuminated region and an adjacent dark region in the detection field of view allows the sample refractive index to be deduced geometrically. Differential refractometers, for example that disclosed in U.S. Pat. No. 5,157,454, have been developed for measuring a difference in refractive index between a test sample and a known reference sample, whereby variable test conditions effecting the measurement result, such as sample temperature, illumination level, etc., can be xe2x80x9csubtracted outxe2x80x9d to yield a more accurate and precise measurement result. The prior art differential refractometers known to applicants involve moving parts which malfunction or wear out over time, and/or are restricted to the transmitted light variety so as to prevent measurement of samples having relatively high opacity.
Optical biosensor devices designed to analyze binding of analyte molecules to a binding layer by observing changes in internal reflection at a sensing interface are also part of the related prior art. More specifically, U.S. Pat. No. 5,313,264 to Ivarsson et al. describes an optical biosensor system that comprises a plurality of side-by-side sensing surfaces 39A-D illuminated by a streak of light 5 extending transversely across the sensing surfaces, and an anamorphic lens system 6 by which rays of light reflected from the respective sensing surfaces are imaged on corresponding columns of a two-dimensional array 7 of photosensitive elements. Accordingly, the signals from the photosensitive elements can be processed to determine a minimum reflectance associated with the resonance angle at each sensing surface. Although the system described in U.S. Pat. No. 5,313,264 avoids the use of moving parts, it is nevertheless optically complex and requires a two-dimensional array, factors that are accompanied by an increase in cost.
Finally, it is noted that one-dimensional (linear) arrays of photosensitive elements cells are commonly used in automatic refractometers designed to take non-differential readings with respect to a single test sample. Examples can be found in U.S. Pat. No. 4,640,616 (Michalik) and U.S. Pat. No. 6,172,746 (Byrne et al.). However, applicants are unaware of any critical angle optical device for differential refractive index measurements that operates using a linear array, despite the recognized economy offered by this type of array.
Therefore, it is an object of the present invention to provide an optical configuration for differential refractive index measurements wherein a first sample and a second sample are illuminated by a single illuminating beam.
It is another object of the present invention to provide an optical configuration for differential refractive index measurements that does not rely on moving parts.
It is a further object of the present invention to provide an optical configuration for differential refractive index measurements wherein detected light has been reflected rather than transmitted at an optical interface of the configuration.
It is a further object of the present invention to provide an optical configuration for critical angle differential refractive index measurements wherein light interacting at first and second optical interfaces corresponding to a first sample and a second sample is detected by a single linear scanned array of photoelectric cells.
It is a further object of the present invention to provide an optical configuration for differential refractive index measurements in accordance with the objects stated above, and which operates based on surface plasmon resonance principles for use in a biosensor device.
An optical configuration formed in accordance with a first embodiment of the present invention comprises an optical path defining a meridional plane of the configuration. A high index prism in the optical path includes a sample surface divided by a partition residing in the meridional plane, such that a first sample and a second sample supported by the sample surface are located on opposite sides of the meridional plane to establish a first optical interface associated with the first sample and a second optical interface associated with the second sample. An illumination beam traveling along the optical path illuminates both optical interfaces simultaneously to provide a first partial beam defined by the refractive index of the first sample and a second partial beam defined by the refractive index of the second sample. A collecting lens collimates the first and second partial beams and an optical multiplexor means receives the collimated partial beams and defines first and second optical channels containing optical signal information associated with the first and second partial beams, respectively. A cylinder lens and/or a biprism after the optical multiplexing means deflects the partial beams laterally toward the meridional plane of the system for illuminating a linear scanned array of photoelectric cells aligned in the meridional plane. In a first embodiment, the optical multiplexing means comprises a liquid crystal shutter programmed to alternately transmit one partial beam while blocking the other partial beam such that a given scan of the linear array provides signal information with respect to either the first partial beam or the second partial beam, depending upon the corresponding state of the liquid crystal shutter. In a second embodiment, a similar arrangement is used, however the liquid crystal shutter is programmed to transmit and block predetermined portions of each partial beam in an alternating fashion, such that for a given scan of the linear array some of the array cells will provide signal information relating to the first partial beam and some of the cells will provide signal information relating to the second partial beam. The first partial beam exhibits a feature, such as a shadowline or resonance minimum, on the linear scanned array the location of which is indicative of the refractive index of the first sample, while the second partial beam exhibits a similar feature the location of which is indicative of the refractive index of the second sample.
A third embodiment based on the first and second embodiments is an adaptation of the basic configuration in order to observe molecular interactions, particularly specific binding of analyte molecules to a binding layer, using the principles of surface plasmon resonance. In accordance with the third embodiment, a thin metallic film is applied to a slide placed on the sample surface or directly to the sample surface, and the first sample and second sample are brought into contact with the metallic film to define first and second evanescent wave optical interfaces. In this embodiment, the locations of resonance minimums exhibited by the first and second partial beams are detected.
The present invention further encompasses methods for measuring a difference in refractive index between a first sample and a second sample based on the specified optical configurations.